KR950007222B1 - Production of l-leucine by microorganism cultivation - Google Patents

Abstract

L-leucine is produced by i) treating corynebacterium donga MS-03178 (KFCC 10011) with UV, NTG or DFS to obtain the mutant, corynebacterium donga MS-03301 (KCTC-8507P), ii) innoculating the obtd. mutant into a medium and fermenting at 30 deg.C and 120 rpm for 20 hrs. to obtain the starter, and iii) innoculating the obtd. starter into the medium contg. saccharide, and fermenting at specific growth rate 3.5-5.5 g/l/hr., pH 6.5-7.5 and aeration rate 0.1-1.0 vvm. The obtd. L-leucine is useful as medicine, food additive etc..

Description

Production of L-Leucine by Microbial Fermentation

The present invention relates to the production of L- leucine by microbial fermentation.

L-leucine is an essential amino acid, which is used as a pharmaceutical, food additives, etc., and is recently attracting attention as a raw material and additive of liquid crystals or waste oil decomposition agents.

The present invention relates to a method for improving the production yield of L- leucine by using "mutant strains in which the logarithmic growth group is significantly longer than the parent strain" when producing L- leucine by microbial fermentation.

In other words, when L-leucine is produced by microbial fermentation, the growth of bacteria is not maintained as usual, and the specific growth rate of bacteria is 3.5-5.5g from 10 hours after the inoculation of the bacteria to the end of fermentation. It relates to a method that can significantly improve productivity by separating and using bacteria which maintain a constant growth in / L / H.

The production method of L-leucine known so far is mainly derived from wheat gluten and other protein hydrolysates. However, "fermentative production using microorganisms" uses L-isoleucine-required strains from Cerachia malceseus. Method (Japanese Patent Digestion 47-26313), Pseudomonas genus, Agrobacterium genus, Escherichia coli, Bacillus genus, etc. A method of cultivating one or two kinds of bacteria (Japanese Patent Digestion 52-64486), L-Aso Method using a strain that requires leucine, L-threonine, and L-methionine as growth factors (US Pat. No. 14964 0), S- (β-aminoethyl) -L-cysteine resistant strain in a medium containing acetic acid Method of accumulating L-leucine by culturing (Japanese Patent Digestion 51-37347), Method of increasing L-leucine accumulation by controlling oxygen supply condition in culture of L-leucine producing strain (Japanese Patent Digestion 57-798), in culture L-bar (Japanese Patent Digestion 56-127095), semi-continuous fermentation method (Korean Patent Publication No. 83-2485), which adds additional medium periodically or intermittently to maintain the sugar concentration in the medium. It is well known.

In the production of amino acids by fermentation industry using general microorganisms, especially in the production of amino acids, the growth of bacteria is usually divided into four sections. First, the bacteria are changed in the lag phase after inoculation of the bacteria into the medium. There are steps to adapting to the environment and preparing for division and growth.

This section usually takes as little as 2-5 hours and as long as 10-20 hours, depending on the type of bacteria and the condition of the medium. The consumption of nutrients in bacteria is gradually increased, but the accumulation of target products is hardly observed. (1 part of Figure I)

The next stage is the logarithmic phase, where bacterial division and growth (strain growth and volume growth) are maximized, which rapidly absorbs and consumes nutrients in the medium and increases the exponential growth of the bacterial count due to cell division. Of course, the increase in volume also appears at the same time.

In general, after this period, microorganisms appear to accumulate the most desired products.

In other words, the strains that have grown after somatic growth have reached the maximum capacity (synthesis) to synthesize products, and accumulate or discharge a large amount of the target product in and out of the body.

The next step is to enter into a state in which the growth of the bacteria is stopped, ie, the equilibrium between division, volume increase and death.

At the beginning of this period, the specific growth rate of bacteria is significantly reduced, while the specific production rate and consumption rate remain almost the same as log growth.

This is an excellent time to biosynthesize the desired product as the germ breakdown and body growth decrease, and a large amount of product is accumulated.

In general, however, this period appears briefly and thereafter, the specific production rate and the specific consumption rate decrease gradually. This phenomenon is caused by the deterioration of the bacteria as a result of the deterioration of the activity as time passes, and the accumulation of the target product in vitro. It has been interpreted that (final product) appears by inhibiting or suppressing the biosynthetic action of bacteria.

At the beginning of this period, the consumption trend of nutrients tended toward the biosynthesis of the target product rather than the growth of the bacteria, but afterwards, the specific growth rate of the bacteria was close to “0” at this time, and the specific consumption rate of nutrients (sugar) Also, compared with logarithmic steam, the accumulation of product per unit time, that is, the specific production rate, is also reduced. (Part 3 of Figure I)

Afterwards, bacteria were killed due to their altered external environment, namely, internal and external accumulation or increase of inhibitors such as excretory substances and toxins, and decrease in the concentration of available nutrients. Enter the death phase that appears.

In this period, the germ is almost lost in growth and growth capacity, and the biosynthetic function of the target product is so low that the economic value is lost.

As described above, the bacterium life cycle of the general bacteria in the batch culture (Fed Batch culture) shows the same tendency, although there is a time difference between the growth and growth of the bacteria.

The present inventors have been studying the production of L- leucine by microbial fermentation for many years, and by separating and using the mutant strain (see Fig. Ll) which is different from the growth curve of the general microorganism as described above, L- leucine is remarkable in the culture medium. It is to find out what can be accumulated to complete the present invention.

In other words, L-leucine-producing strain Corynebacterium Dong-A MS-03178 (KFCC-10011) strain and its mutant strains when producing L- leucine, as can be seen in the growth curve of the bacteria as the maximum capacity for the production of bacteria In other words, when using a strain or a method in which the logarithmic multiplier with the highest specific production rate is extended compared to the parent strain, the accumulation of L-leucine in the culture medium can be maximized. Isolation of L-leucine by the conventional method by separating and using the strain Corynebacterium sinus MS-03301 (KCTC-8507p) maintained at a constant growth rate of 3.5-5.5 g / L / H (see Table I and Figure III). Production can be significantly increased to complete the present invention.

In other words, the production of L- leucine by most of the conventional microbial fermentation method as shown in Figure I to secure the bacteria at a light concentration in the pre-fermentation fermentation and one cycle of culture according to the general "cell growth curve" but the present invention It is an innovative method that uses "strains that show linear growth" by continuing logarithmic growth and straightening cell division and volume growth.

The strain used in the present invention is a common strain of Corynebacterium Dong-a MS-03178 (KFCC-10011), that is, UV irradiation or NTG (N-methyl-N'-methyl-N-nitrosoguanidine), Mutant strains treated with chemically modified organic agents such as DFS (diethyl sulfate) were used separately.

The strain strain of Corynebacterium genus MS-03301 (KCTT-8507p) of the present invention was identified as strain characteristics according to Burgess manual is as follows.

1. Morphological Properties

1) Shape: Bacillus

2) Size: 1 2 × 4.8μ

3) Gram Dyeing: Positive

4) Mobility: None

5) Poca: No Formation

2. Cultural Properties

1) Juicy Agar Slope Cultivation: (30 ℃ × 48 hours)

2) Juicy agar plate culture: growth good, round

3) Juicy Agar Subscript Culture: Growing Rough

4) Gelatin Culture: Slightly Soluble

3. Physiological Properties

1) Growth temperature: 20 ℃ -40 ℃

2) Optimum temperature for growth: 30 ℃ ± 1 ℃

3) Growth pH: 45-9-5

4) Optimal pH for growth: 6.5-7.5

5) Oxygen Requirements: Aerobic

6) Death temperature: 70 ℃ × 15 minutes

7) Litmus milk: dyed purple

8) Indole Skidol Generation: Voice

9) Nitrate Reducibility: None

10) Hydrogen Emulsion Production: Negative

11) Catarase Production: Positive

12) Methylene Blue Reaction: Positive

13) Sugar Fermentation

Glucose: Yes

Sugar: Yes

Fructose: Yes

Raffinose: Indistinguishable

Mannos: In

Galactose: None

Casiros: Indistinguishable

Starch: no

Inulin: None

Inositol: None

14) Nutritional composition: thiamine, isoleucine

The strain specificity of the strain Corynebacterium Dong-a MS-03301 (KCTC-8507p) used in the present invention cannot accurately determine the effect of leucine biosynthesis and the effect of promoting biosynthesis. It is thought that this is directly related to the extension of the logarithmic generation period that can accumulate the most target product.

In the present invention using the above strain, sugars such as molasses, starch hydrolyzate, glucose and fructose may be used as the carbon source in the medium, and as nitrogen sources, nitrogen compounds such as ammonium sulfate, urea, ammonia, peptone, yeast extract, Meat extract, corn steep liquor, soybean meal hydrolyzate, casein hydrolyzate and the like can be used. As inorganic substances, potassium phosphate dibasic, potassium diphosphate, ferrous sulfate, magnesium sulfate, manganese sulfate and the like can be used.

The culture method is incubated for 40 hours in aerobic temperature while maintaining the pH at 6.5-7.5 within the fermentation temperature of 30 ℃.

L-leucine accumulated in the culture complete solution is adsorbed and separated from the ion exchange resin according to a conventional method to obtain an eluent, and depurified and concentrated to obtain purified L-leucine crystals.

Drawing. I: Proliferation Curve of L-Leucine Fermentation by Normal Strains

① LAG PHASE

② LOGARITHMIC PHASE

③ STAT10NARY PHASE

④ DEATH PHASE

Drawing. II: Proliferation Curve of L-Leucine Fermentation by Strains of the Invention

① LAG PHASE

② LOGARITHMIC PHASE

TABLE 1

Drawing. III: Comparison of Dry Cell Mass by Elapsed Time between KFCC-10011 and KCTC-8507P

(Reference) Dry cell weight measurement method:

Centrifugal separation (3,000g × 20min) → Cell washing (IN HCl) → Centrifugal separation (3,000g × 20min) → → vacuum drying (12hr × 55 ℃) → basis weight

Example 1

Use strain: Corynebacterium dongah MS-03301 (KCTC-8507P)

Species Basis

Glucose 6.0% Sodium Chloride 0.25%

Polypeptone 1.0% Biotin 50μg / L

Meat extract 0.5% Urea 0.4%

Yeast Extract 0.5%

Corn Dipping 0.6%

(CSL)

Fermentation medium composition:

Glucose 12% Potassium Phosphate 0.06%

Corn Dipping 8% Potassium Diphosphate 0.02%

(CSL) Biotin 50μg / L

Ammonium Sulfate 0.1% Thiamine Hydrochloride 2mg / L

Ferrous Sulfate 0.001% L-Isoleucine 500μg / L

Manganese Sulfate 0.001% Calcium Carbonate (Additional Sterilization) 5%

Ammonium acetate 0.3% antifoam

Magnesium sulfate 0.1%

Culture method:

50 ml of the seed medium adjusted to pH 7.2 of the above medium composition was dispensed into a 500 ml shake flask, autoclaved and cooled at 120 ° C. for 10 minutes, and then the new strain MS-03301 (KCTC-8507P) of the present invention was diluted with platinum. Inoculated and shaken at 30 ° C. for 20 hours at 120 revolutions were used as the seed culture.

300 ml of the fermentation broth with pH adjusted to 7.0 was dispensed into a 1 L Erlenmeyer flask, autoclaved at 120 ° C. for 15 minutes, cooled, inoculated with 12 ml of the prepared seed culture medium, and incubated at 30 ° C. for 40 hours.

After completion of the culture, L- leucine production in the culture medium was 31g / L and in this example, the average specific production rate of the new strain was 0.775g / L / H.

Example 2

In the fermentation broth of Example 1, 2 L of each medium except calcium carbonate was injected into a 5 L small fermentation tank, and autoclaved at 120 ° C. for 15 minutes, and then cooled.

Inoculate 80 ml each of the previously prepared spawn culture solution and incubate at 30 ° C. for 40 hours under conditions of 600 rpm and 1 vvm.

pH was maintained at 6.5, 7.0, 7.5 and 8.0, respectively.

After completion of fermentation, L-leucine production and average specific production rate in the culture are shown in Table 2 below.

TABLE 2

Example 3

Aeration for the culture was 0.5, 1.0, 1.2, 1.5vvm, respectively, pH was maintained at 7.0. Other culture methods are carried out in the same manner as in Example 2.

After completion of fermentation, L-leucine production and average specific production rate in the culture are shown in Table 3 below.

TABLE 3

Claims (4)

Nutrient containing sugar as the main ingredient of Corynebacterium Dong-a MS-03301 (KCTC-8507P), a strain in which the logarithmic growth rate, which is the most active in the production of L-leucine by microorganisms, is longer than that of the parent. A method for producing L-leucine by a microorganism, characterized by significantly increasing the production accumulation of L-leucine by culturing aerobicly in a medium. The method according to claim 1, wherein the non-proliferative culture of the cells is incubated at 3.5-5.5 g / L / H. The method of claim 1, wherein the culture is maintained at a pH of 6.5-7.5. The method according to claim 1, wherein the culture is carried out while maintaining the aeration amount at 0.5 to 1.0 vvm.